The effect of anti-CD13 mAb in adhesion depends on CD13 cross-linking. (A) U-937 cells were incubated for 30 min with anti-CD13 mAb 452, its F(ab)′₂ or Fab fragments, or with the anti-CD13 mAb WM-4.7 alone or followed by a 30-min incubation with F(ab)′₂ fragments of a GAM antibody, all at 1.0 μg/ml. Fragments of the 452 mAb showed similar binding capacity as the complete mAb (IgG), as determined by FACS (inset). (B) Confocal imaging of U-937 cells treated with FITC-conjugated, anti-CD13 mAb WM-4.7 (1.0 μg/ml, upper left) or Texas-Red™-conjugated mAb 452 (1.0 μg/ml, lower left) for 15 min at 37°C. EOMA cells stably expressing hCD13-GFP were imaged before (upper right) or after (lower right) treatment with the mAb 452 (1.0 μg/ml) for 15 min at 37°C. (C) U-937 cells were incubated with UV-inactivated HCoV-229E for 1 h at 37°C at doses corresponding to the indicated multiplicity of innoculation. Unbound virus was washed away, and cells were allowed to adhere to HUVEC for 20 min at 37°C. Arithmetic mean and sd of three independent experiments are shown. *, P < 0.05, unpaired Student’s t-test between the individual treatment condition and the control, untreated condition in each panel.

CD13 is present in filopodia at the zones of contact, and its expression in the monolayers is required for mAb-treated monocytic cells to adhere. (A, Left) EOMA cells stably expressing GFP-CD13 and grown at subconfluency were imaged by confocal microscopy. A single angle of the 3-D projection shown in Supplemental Video 1 is shown. (A, Center) GFP-CD13-expressing EOMA cells were imaged after they had established a monolayer. (A, Right) Anti-CD13, mAb-treated (1.0 μg/ml), calcein red-labeled U-937 cells were allowed to adhere onto CD13-GFP-expressing C33A cells and imaged by time-lapse confocal microscopy. As soon as the monolayers started projecting filopodia toward the adhering monocytic cells, cells were fixed, and 3-D reconstructions were generated as described in Materials and Methods. The upper images represent single rotation angles from the projections shown in Supplemental Videos 2 and 3, respectively. The lower panel shows a single angle from the projection shown in Supplemental Video 4, where a monocytic cell is adhering to a single C33A cell. (B) HUVEC, U-937 cells, or both (as indicated in the schematic) were preincubated with 452 mAb (1.0 μg/ml). After washing, U-937 cells were allowed to adhere for 15 min. The graph represents the arithmetic mean and sd of five independent experiments. (C) U-937 cells were preincubated with anti-CD13 mAb (Ab) or without (no Ab) for 30 min at 37°C and allowed to adhere onto monolayers of nontransfected (NT), vector-transfected (VECTOR), or V5-tagged, hCD13-transfected C33A cells in the absence (hCD13) or presence (hCD13/Ab) of excess anti-CD13 mAb. Each bar represents the arithmetic mean and sd of three independent experiments.

CD13 ligation induces monocytic cell adhesion to endothelial monolayers. (A) Three-dimensional (3-D) reconstructions of calcein red-labeled HUVEC monolayers untreated (middle) or treated with anti-CD13 mAb (1.0 μg/ml, bottom) after adhesion of calcein green-labeled U-937 cells. The top images show HUVEC monolayers without U-937 cells to demonstrate the absence of fluorescence bleed-through between channels. The right panels represent the green channel-only images at the plane of focus of the monocytic cells. Images representative of three independent experiments are shown. (B) C33A cells expressing V5-tagged CD13 were incubated with anti-CD13 (452; 1.0 μg/ml) or anti-V5 mAb at the doses indicated and washed, and monocytic cells were allowed to adhere for 15 min. Adhesion was quantified by fluorometry, as described in Materials and Methods. The graph is representative of the arithmetic mean and the sd of two independent experiments. (C) HUVEC monolayers were preincubated with the indicated anti-CD13 mAb (1.0 μg/ml), an isotype-matched IgG, or the CD13 inhibitor bestatin (100 μM). After washing, U-937 cells were allowed to adhere for 15 min. The percentage of adherent cells as compared with untreated cells [control (Ctrl)] is shown. The graph represents the arithmetic mean and sd of six independent experiments. (D) The same experiment as in C but preincubating U-937 cells with the indicated mAb. The graph represents the arithmetic mean and sd of five independent experiments. (E) PBMC (left) or HUVEC (right) were incubated with 452 mAb for 30 min and 1 h, respectively. Adhesion was quantified as above. *, P < 0.05, unpaired Student’s t-test between the individual treatment condition and the control.

CD13 directly participates in the adhesion process. (A, Upper panel) U-937 cells were preincubated with biotinylated anti-CD13 mAb for 30 min and allowed to adhere onto C33A cells nontransfected or transfected with the indicated construct, including a V5-tagged (•), htCD13. After 20 min, all cells (C33A plus nonadherent and adherent U-937 cells) were pelleted and lysed. CD13 was immunoprecipitated with streptavidin-agarose and proteins resolved by SDS-PAGE. The coimmunoprecipitation of the V5-tagged CD13 from the monolayers was assayed by an immunoblot anti-V5 (V5). (A, Lower panel) The total amount of monocytic CD13 immunoprecipitated from each sample with the biotinylated mAb was detected with streptavidin-HRP (CD13). (B) U-937 cells were treated with the anti-CD13 mAb (1 μg/ml) for 30 min at 37°C. After extensively washing the excess antibody, the indicated doses of sCD13 were added and incubated with the cells for a further 30 min at 37°C. After extensive washing of the unbound protein, cells were allowed to adhere onto HUVEC in the absence (black bars) or presence (gray bars) of the same dose of the recombinant protein added immediately before the assay. The graph represents the arithmetic mean and sd of two independent experiments. (C) The same experiment as in B was conducted but preincubating HUVEC with anti-CD13 mAb. After extensive washing, sCD13 was added and incubated for 30 min at 37°C. The protein was washed away, and monocytic cells were allowed to adhere in the absence (black bars) or presence (gray bars) of the same dose of protein added before the assay. (D) Untreated (no Ab) or anti-CD13 mAb-treated U-937 cells (Ab) were allowed to adhere onto the wells of a plate coated with the indicated amounts of sCD13 as described in Materials and Methods. The graph represents the arithmetic mean and sd of two independent experiments normalized against the control of BSA-coated wells as determined by fluorometry. *, P < 0.05, unpaired Student’s T-test between indicated conditions (B, C) or untreated control (D).

CD13-induced adhesion is signal transduction-dependent. (A) U-937 or HUVEC cells were preincubated with herbimycin or its vehicle DMSO (CTRL) for 2 h at 37°C (U-937+HERB) and (HUVEC+HERB), respectively. After washing three times, the anti-CD13 mAb 452 was added to U-937 cells (U-937 Ab) or HUVEC (HUVEC Ab) and incubated for 30 min at 37°C in the continued presence of the inhibitor or the vehicle. After three washes, U-937 cells were allowed to adhere for 15 min. Graphs represent normalized data from three independent experiments. (B) HUVEC were preincubated (PRE) with cytochalasin-D 200 nM for 30 min at 37°C before addition of the anti-CD13 mAb (Ab), and adhesion was allowed to proceed in the presence of the same concentration of cytochalasin-D (Cyt) or an equivalent volume of vehicle (V). Two extra samples were included (Lanes 4 and 5), in which U-937 cells had been pretreated with cytochalasin-D 2 μM (U-937-Cyt) for 30 min at 37°C before the assay. The graph represents the arithmetic mean and sd of two independent experiments. (C) U-937 cells were preincubated with 2 μM cytochalasin-D or the vehicle. After 30 min at 37°C, the anti-CD13 mAb 452 was added (Ab) in the continued presence of the vehicle or cytochalasin. Finally, cells were washed five times and allowed to adhere to HUVEC for 15 min (Adhesion) in the presence of vehicle or cytochalasin-D at the same concentration. In Lanes 4 and 5, mAb-treated (1.0 μg/ml) U-937 cells preincubated with cytochalasin or vehicle were allowed to adhere onto cytochalasin-treated HUVEC (HUV Cyt). Adhesion was quantified as above. (D) Control U-937 cells (no Ab) or cells treated with the anti-CD13 mAb 452 for 15 min at 37°C (Ab) were fixed, permeabilized, stained with tetramethyl rhodamine isothiocyanate-conjugated phalloidin, and analyzed by confocal microscopy.

CD13 ligation impairs transendothelial migration of leukocytes in vivo. (A) The total number of leukocytes was determined in the peritoneal lavage of animals injected with hamster anti-mouse CD13 mAb K1 (anti-CD13), hamster IgG (IgG), or the vehicle (PBS) in a peritonitis model as described in Materials and Methods. Each bar represents the arithmetic mean and sd of the number of leukocytes from at least 10 mice. (B and C) H&E staining of sections of peritoneal walls from an IgG- and an anti-CD13-treated animal. (D) Determination of the relative number of mononuclear (MN) versus polymorphonuclear (PMN) cells was performed by microscopic analysis of peritoneal cells in at least three fields per sample from at least three mice per group. (E) Determination of the relative number of monocytes (CD11b+/GR1–) versus polymorphonuclear (CD11b/Gr1 double-positive) cells in the circulation was performed by flow cytometric analysis of the peripheral blood from at least three mice per group. *, P < 0.05, unpaired Student’s t-test.